Speaker: TzeFeng-Tian Advisor: Ruey-Shyang,Hseu, PhD

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Diego Martinez, Randy M Berka, Bernard Henrissat, Markku Saloheimo, Mikko Arvas, Scott E Baker, Jarod Chapman, Olga Chertkov, Pedro M Coutinho, Dan Cullen, Etienne G J Danchin, Igor V Grigoriev, Paul

Robbertse, Asaf A Salamov, Monika Schmoll, Astrid Terry, Nina Thayer, Ann Westerholm-Parvinen, Conrad L Schoch, Jian Yao, Ravi Barabote, Mary Anne Nelson, Chris Detter, David Bruce, Cheryl R Kuske,

1 Diego Martinez, Randy M Berka, Bernard Henrissat, Markku Saloheimo, Mikko Arvas, Scott E Baker, Jarod Chapman, Olga Chertkov, Pedro M Coutinho, Dan Cullen, Etienne G J Danchin, Igor V Grigoriev, Paul Harris, Melissa Jackson, Christian P Kubicek, Cliff S Han, Isaac Ho, Luis F Larrondo, Alfredo Lopez de Leon, Jon K Magnuson, Sandy Merino, Monica Misra1, Beth Nelson, Nicholas Putnam, Barbara Robbertse, Asaf A Salamov, Monika Schmoll, Astrid Terry, Nina Thayer, Ann Westerholm-Parvinen, Conrad L Schoch, Jian Yao, Ravi Barabote, Mary Anne Nelson, Chris Detter, David Bruce, Cheryl R Kuske, Gary Xie, Paul Richardson, Daniel S Rokhsar, Susan M Lucas, Edward M Rubin, Nigel Dunn-Coleman, Michael Ward & Thomas S Brettin. Nature Biotechnology 26: , Speaker: TzeFeng-Tian Advisor: Ruey-Shyang,Hseu, PhD 1

2 Many evidences show the importance of bioethanol Reduce dependent on imported petroleum Lower environmental pollution Cellulosic ethanol offers large reductions in green house gas(ghg) emissions. 2

3 However, The high cost of hydrolyzing biomass polysaccharides to fermentable sugars Image Source: 3

4 Objective Improve the productivity of enzyme Reduce the cost of process 4

Bioethanol process overview Cellulase Hemicellulase

Source: CornStover: http://blogs.knoxnews.

5 Bioethanol process overview Cellulase Hemicellulase Pectin-degrading enzyme straw corn stover grass Image Source: CornStover: Grass: Straw: FlowChart refer from: Jeffrey S.Tolan, Iogen s process for producing ethanol from cellulosic biomass, Clean Techn Environ Policy 3 (2002)

Several organisms used in the production of cellulases in

(fungi) Penicillium (fungi) Trichoderma (fungi) Image Source:

fr/enseigne/biotech/galerie/champignons/tableau/champignons.

37/~bernie/sciproject/project/kingdoms/fungi6/aso.

6 Several organisms used in the production of cellulases in industry: Humicola (plantae) Bacillus (bacteria) Aspergillus (fungi) Penicillium (fungi) Trichoderma (fungi) Image Source: Humicola, Aspergillus: Penicillium: Bacillus: Trichoderma: 6

7 7

8 Trichoderma reesei Mesophilic filamentous fungus asexual of Hypocrea jecorina Mutant strain derived from T. viride QM6a during 2 nd War World isolated Trichoderma reesei Trichoderma was Isolated during World war II in rotted U.S. Army cotton tents in the South Pacific. Image Source: Trichoderma: 8

9 Scheme for the usage of T.reesei Step I (C 6 H 12 O 5 )n Cellulose + nh 2 O Water Trichoderma Cellulase nc 6 H 12 O 6 Glucose Step II Glucose Yeast 2 C 2 H 5 OH ethanol + 2CO 2 Carbon dioxide Sources: Ramesh Maheshwari, Genome sequence of a fungus for the biofuel industry, CURRENT SCIENCE, VOL. 95, NO. 2, 25 JULY

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11 ab initio methods: Ab initio quantum chemistry methods are computational chemistry methods based on quantum chemistry Definition refer to: 11

Libraries used for genome sequencing JGI: Department of Joint

Carolina State University Shotgun Sequencing Genome Database s

9Mb (89 scaffolds + 97 contigs) Represents more than 99% of the

12 Libraries used for genome sequencing JGI: Department of Joint Genome Institute NCSU: Fungal Genomics Laboratory, North Carolina State University Shotgun Sequencing Genome Database s Phred/Phrap/ Consed (Computer Software) 33.9Mb (89 scaffolds + 97 contigs) Represents more than 99% of the genome Image Source: Trichoderma: 12

13 Organism Type Genome size(mb) Number of genes predicted Year of completion Aspergillus niger Fungus , Fusarium graminearum Fungus Aspergillus fumigatus Fungus , Aspergillus nidulans Fungus 30 9, Aspergillus oryzae Fungus 37 12, Cryptococcus (Filobasidiella) neoformans Fungus 20 6, Magnaporthe grisea Fungus , Ashbya gossypii Fungus 9.2 4, Candida glabrata Fungus , Debaryomyces hansenii Yeast , Kluyveromyces lactis Yeast 10~12 5, Yarrowia lipolytica Yeast 20 6, Neurospora crassa Fungus 40 10, Schizosaccharomyces pombe Yeast 14 4, Encephalitozoon cuniculi Microsporidium 2.9 1, Saccharomyces cerevisiae Baker's yeast , Table The history of Genome Sequencing in fungi 13

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15 RESULTS Features of T.reesei genome Conserved synteny in T.reesei Protein domains in T.reesei Carbohydrate-active enzymes in T. reesei and other fungi Protein Secretion CAZyme gene clusters in T.reesei 15

16 Features of T.reesei genome (1/3) 16

RIP (Repeat-induced point mutation) Repeat-induced point mutation A process that efficiently detects and mutates duplicated sequences. Act only during Sexual Cycle.

17 RIP (Repeat-induced point mutation) Repeat-induced point mutation A process that efficiently detects and mutates duplicated sequences. Act only during Sexual Cycle. ~30% of C:G -> T:A RIP-mutated sequences are also frequent targets for DNA methylation gene silencing RIP illustrates the extraordinary extent to which genomes defend themselves against mobile genetic elements. Figure. The Repeat-induced point mutation process in Neurospora crassa. Reference: James E.Galagan and Eric U. Selker, RIP: the evolutinary cost of genome defense. TRENDS in genetics Vol.20 No.9 September

18 Features of T.reesei genome (2/3) Some repetitive sequences similar to class I & II transposable elements (~1% of the finished genome) contains multiple stop codons. No active transposable elements Repeat-induced point mutation (RIP) may have attenuated the spread of transposons. (similar to other Ascomycetes) 18

19 Telomere A telomere is a region of repetitive DNA at the end of chromosomes, which protects the end of the chromosome from destruction. Image Source: Reference: 19

20 Features of T.reesei genome (3/3) Telomeric repeat TTAGGG was found at the ends of 7 scaffolds in the T. reesei genome assembly. Scaffold Scaffold_31 Scaffold_1 Scaffold_46 Scaffold_45 Scaffold_65 Scaffold_64 Scaffold_87 telomere was found in 7 from 89 scaffolds 20

21 RESULTS Features of T.reesei genome Conserved synteny in T.reesei Protein domains in T.reesei Carbohydrate-active enzymes in T. reesei and other fungi Protein Secretion CAZyme gene clusters in T.reesei 21

22 What is Synteny? Synteny describes the physical co-localization of genetic loci on the same chromosome within an individual or species The synteny maps of chromosomes of human & mouse. Image Source: Reference: 22

23 Conserved synteny in T.reesei Adaptation of organism Prone to frequent insertion, duplication, chromosomal breaks Syntenic blocks mapped to the Trichoderma reesei genome from Fusarium graminearum and Neurospora crassa. LG: linkage group Chr: Chromosome 23

24 RESULTS Features of T.reesei genome Conserved synteny in T.reesei Protein domains in T.reesei Carbohydrate-active enzymes in T. reesei and other fungi Protein Secretion CAZyme gene clusters in T.reesei 24

25 Number of occurences >4 25

26 Protein domains in T. reesei Number of occurences >4 26

27 T.reesei Number of occurences >4 27

28 Protein domains in T. reesei T. reesei lacks several protein families: Pectate lyases, pectin esterases, tannase, feruloyl esterase family members Suggests that the diversity of hemicellulases & pectin-degrading enzyme of T. reesei is relatively small. 28

29 RESULTS Features of T.reesei genome Conserved synteny in T.reesei Protein domains in T.reesei Carbohydrate-active enzymes in T. reesei and other fungi Protein Secretion CAZyme gene clusters in T.reesei 29

30 What is CAZymes? CAZymes: Carbohydrate-active enzymes carbohydrate-binding module glycoside hydrolase glycosyltransferase polysaccharide lyase carbohydrate esterase 30

31 Carbohydrate-active enzymes in T. reesei and other fungi(1/5) division class GH: glycoside hydrolase GT: glycosyltransferase CBM: carbohydrate-binding module CE: carbohydrate esterase PL: polysaccharide lyase Highest in red Lowest in blue 31

cellulases A mixture of 3 types of enzyme [2] : Cellobiohydrolase (CBH): CBHI, CBHII cellulose

cellulose, lichenin and cereal β-d-glucans Beta-glucosidase(BG) Hydrolysis of terminal,

Tolan, Iogen s process for producing ethanol from cellulosic biomass, Clean Techn Environ Policy 3

KENT KIRK1* The Cellulases Endoglucanase I and Cellobiohydrolase II of Trichoderma reesei Act

32 cellulases A mixture of 3 types of enzyme [2] : Cellobiohydrolase (CBH): CBHI, CBHII cellulose 1,4-β-cellobiosidase Endoglucanase(EG) Endohydrolysis of (1 4)-β-D-glucosidic linkages in cellulose, lichenin and cereal β-d-glucans Beta-glucosidase(BG) Hydrolysis of terminal, non-reducing β-d-glucosyl residues with release of β-d-glucose Reference: 1. Jeffrey S.Tolan, Iogen s process for producing ethanol from cellulosic biomass, Clean Techn Environ Policy 3 (2002) KAREN M. KLEMAN-LEYER,1 MATTI SIIKA-AHO,2 TUULA T. TEERI,2 AND T. KENT KIRK1* The Cellulases Endoglucanase I and Cellobiohydrolase II of Trichoderma reesei Act Synergistically To Solubilize Native Cotton Cellulose but Not To Decrease Its Molecular Size, APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Aug. 1996, p

33 Catalytic sites of cellulases breaks internal bonds to disrupt the crystalline structure Endoglucanase(EG) Cellobiohydrolase (CBH) two main types: Reducing end Non-reducing end Reference: Beta-glucosidase(BG) hydrolyses the exo-cellulase product into individual monosaccharides 33

34 Carbohydrate-active enzymes in T. reesei and other fungi(2/5) 34

35 hemicellulases Hemicellulose comprises a diverse group of complex polysaccharides The complete degradation requires an arsenal of enzymes. T.reesei has only 16 hemicellulases. xylose glucose mannose galactose arabinose hemicellulase 35

36 Carbohydrate-active enzymes in T. reesei and other fungi(3/5) xylanase α L-arabinofuranosidase / β-xylosidase α-l-fucosidase 36

37 Pectin-degrading enzymes Pectin is a structural heteropolysaccharide contained in the primary cell walls of terrestrial plants. pectin-backbone α-(1-4)-linked D-galacturonic acid. Image source: 37

38 Carbohydrate-active enzymes in T. reesei and other fungi(4/5) Polygalacturonase, rhamnogalacturonase 38

39 Carbohydrate-active enzymes in T. reesei and other fungi(5/5) 39

40 CAzyme families having a statistically different in T.reesei compare to other fungal genome. 40

41 Enzyme encoded in the specific region Family Enzyme encoded No. of entries GH 92 α-1,2-mannosidase 7 GH 27 α galactosidase 8 GH 95 α 1,2-L-fucosidase 4 GH 64 β-1,3-glucanase 3 Table Enzyme encoded in the specific region α galactosidase of T.reesei Image Source: 41

42 RESULTS Features of T.reesei genome Conserved synteny in T.reesei Protein domains in T.reesei Carbohydrate-active enzymes in T. reesei and other fungi Protein Secretion CAZyme gene clusters in T.reesei 42

43 Protein Secretion (1/3) Homologs of proteins that function in S.cerevisiae is found in T.reesei genome. Have 3 proteins closest homologs in yeast (protein disulfide isomerase, Pdi1p) ER-associated degradation(erad) pathway seems to be more redundant, two orthologs (DER1, UFD) of yeast were found. Proteins involved in protein trafficking can be found. Saccharomyces cerevisiae (species of budding yeast) 43

44 Protein Secretion (2/3) 44

45 Protein Secretion (3/3) The t-snare protein Sso1p of yeast, a receptor for the secretory vesicles on plasma membrane. Has two homologs in T.reesei, then two Sso1 homologs have divergent functions The membrane trafficking system in T.reesei is more diverse than that in S.cerevisiae. 45

46 RESULTS Features of T.reesei genome Conserved synteny in T.reesei Protein domains in T.reesei Carbohydrate-active enzymes in T. reesei and other fungi Protein Secretion CAZyme gene clusters in T.reesei 46

47 CAZyme gene clusters in T. reesei(1/3) Discrete 130/316 (41%) CAZyme genes clusters are found in 25 discrete(noncontinuous) regions. (~2.4Mb, 7% of genome) CAZyme found in the gaps of synteny 47

48 CAZyme gene clusters in T.reesei(2/3) Gene relocation > gene duplication, in responsible for the formation of the CAZyme clusters. Gene relocation Gene duplication 48

49 CAZyme gene clusters in T.reesei(3/3) Swollenin, sequence similarity to the plant expansins, exhibits disruption activity on cellulosic materials 49

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51 Summary 51

52 Future targets Usage of secretion-related gene in other organisms to improve productivity Design mixtures of polysaccharide and lignin-degrading enzymes from different microbial sources for the lignocellulose ethanol biorefineries Genetic engineering of T.reesei genome to enhance its capabilities 52

53 and special thanks to my advisor, Professor Hseu 53

54 Reference 54

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Pathogenic fungi genomics, evolution and epidemiology! John W. Taylor University of California Berkeley, USA

Pathogenic fungi genomics, evolution and epidemiology! John W. Taylor University of California Berkeley, USA Adaptation! Divergence Divergence Adaptation Darwin 1859 Mendel 1866 1830 1850 1870 1890 1910